Abstract:

Cholesterol 7α-hydroxylase（CYP7A1）is a rate-limiting enzyme that breaks down cholesterol into bile acids, which catalyzes cholesterol to produce 7α-hydroxycholesterol, it is an important steroidal drug and intermediate. Taking human CYP7A1 as the research object, firstly, recombinant Pichia pastoris/pPIC3.5K-CYP7A1 was constructed, and then the yield of the target product 7α-hydroxycholesterol increased by site-directed mutation and adaptation to NADPH cytochrome oxidoreductase（CPR）from different sources. Among them, the mutant G485A increased the yield of 7α-hydroxycholesterol by 8.70%. The co-expression of P. pastoris with five different sources of CPR and CYP7A1 increased the yield of 7α-hydroxycholesterol, and the CPR（SgCPR）derived from Siraitia grosvenorii was the most compatible with CYP7A1, which increased the yield of 7α-hydroxycholesterol by 82.26%. Based on the above research, the co-expression of G485A and SgCPR in P. pastoris increased the yield of 7α-hydroxycholesterol by 133.79%, and the yield was 0.25 mg/L. The human cholesterol 7α-hydroxylase was successfully expressed in P. pastoris, and the yield of 7α-hydroxycholesterol increased based on molecular modification of CYP7A1 and adaptation of CPR from different sources.

Key words: steroidal drugs, cholesterol, cholesterol 7α-hydroxylase, 7α-hydroxycholesterol, Pichia pastoris